Tau protease compositions and methods of use

Abstract

Disclosed are mammalian tau proteases, as well as proteolytically-active fragments, variants, and mutants thereof. Also disclosed are polynucleotides and recombinant expression vectors that encode these polypeptides, as well as methods for producing such proteins in selected recombinant host cells, and for using the compositions in a variety of diagnostic and analytical assays.

What is claimed is: 
     
       1. A method of identifying a serine protease inhibitor, the method comprising contacting a compound suspected of inhibiting serine protease activity with a peptide or polypeptide comprising enhanced or diminished serine protease activity when compared to that of a naturally occurring peptide or a naturally occurring polypeptide, and an amino acid sequence that is (i) at least 95%, (ii) at least 96%, (iii) at least 97%, (iv) at least 98%, or (v) at least 99% identical to (A) an at least 50-amino acid contiguous sequence from SEQ ID NO:6, (B) an at least 60-amino acid contiguous sequence from SEQ ID NO:6; (C) an at least 70-amino acid contiguous sequence from SEQ ID NO:6; (D) an at least 80-amino acid contiguous sequence from SEQ ID NO:6; or (E) an at least 90-amino acid contiguous sequence from SEQ ID NO:6 and wherein the peptide or polypeptide comprises at least one substitution at a position that corresponds to amino acid residue 5, 257, 260, 266, 272, 273, 279, 280, 296, 301, 303, 304, 315, 317, 332, 335, 336, 337, 342, 363, 369, 389, 406 or 427 of SEQ ID NO:6, wherein inhibition of the tau protease activity by the compound is indicative of serine protease inhibitory activity of the compound. 
     
     
       2. A method for producing a truncated tau protease or a tau protease variant, comprising (a) culturing a population of recombinant bacterial host cells comprising a polynucleotide sequence that encodes an isolated peptide or polypeptide, wherein the isolated peptide or polypeptide comprises serine protease activity and an amino acid sequence that is (i) at least 95%, (ii) at least 96%, (iii) at least 97%, (iv) at least 98%, or (v) at least 99% identical to (A) an at least 50-amino acid contiguous sequence from SEQ ID NO:6, (B) an at least 60-amino acid contiguous sequence from SEQ ID NO:6; (C) an at least 70-amino acid contiguous sequence from SEQ ID NO:6; (D) an at least 80-amino acid contiguous sequence from SEQ ID NO:6; or (E) an at least 90-amino acid contiguous sequence from SEQ ID NO:6 under conditions conducive to the expression of the truncated tau protease or the tau protease variant, and (b) recovering the expressed truncated tau protease or the tau protease variant from the population of host cells or the culture medium wherein the truncated tau protease or the tau protease variant is configured to be a drug target, and the truncated tau protease or the tau protease variant comprises enhanced or diminished serine protease activity when compared to that of a naturally occurring tau protease or a naturally occurring tau protease variant, and wherein the tau protease or tau protease variant and isolated peptide or tau polypeptide comprises at least one substitution at a position that corresponds to amino acid residue 5, 257, 260, 266, 272, 273, 279, 280, 296, 301, 303, 304, 315, 317, 332, 335, 336, 337, 342, 363, 369, 389, 406 or 427 of SEQ ID NO:6. 
     
     
       3. The method according to  claim 2  wherein the isolated peptide or polypeptide is (i) about 70 to about 250 amino acids in length; (ii) about 80 to about 220 amino acids in length; or (iii) about 100 to about 210 amino acids in length. 
     
     
       4. The method according to  claim 2  wherein the isolated peptide or polypeptide comprises (i) the amino acid sequence of any one of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5; or (ii) 4R2N, 4R1N, or 4R0N isoforms of tau. 
     
     
       5. A composition comprising a pharmaceutically-acceptable carrier and the tau protease or variant according to  claim 2 . 
     
     
       6. The composition according to  claim 5 , for use in Alzheimer's disease therapy, Alzheimer's disease diagnosis, Alzheimer's disease amelioration of symptoms, or drug discovery for a tauopathy, a neural deficit, dementia, senility, age-related memory loss, or traumatic brain injury. 
     
     
       7. A recombinant serine protease prepared by the method of  claim 2 . 
     
     
       8. The method according to  claim 2  wherein the isolated peptide or polypeptide further comprises a label comprising epitope tags. 
     
     
       9. The method according to  claim 2  wherein the isolated peptide or polypeptide further comprises a label comprising fluorescent protein fusions. 
     
     
       10. The method according to  claim 2  wherein the isolated peptide or polypeptide further comprises a label comprising enzyme tau fusions comprising β galactosidase-tau or tau-β galactosidase. 
     
     
       11. The method according to  claim 2  wherein the isolated peptide or polypeptide further comprises a label comprising tau fused to a protein substrate of tau protease. 
     
     
       12. The method according to  claim 2  wherein the isolated peptide or polypeptide further comprises a label comprising tau fused to subcellular localization signals directing it to organelles or to be secreted. 
     
     
       13. The method according to  claim 2  wherein the isolated peptide or polypeptide further comprises a label comprising tau-tau(n) fusion constructs. 
     
     
       14. The method according to  claim 2  wherein the isolated peptide or polypeptide further comprises a label comprising tau chemically labeled with dyes. 
     
     
       15. The method according to  claim 2  wherein the isolated peptide or polypeptide comprises an at least 100-amino acid contiguous sequence from SEQ ID NO:6. 
     
     
       16. The method according to  claim 2 , wherein the isolated peptide or polypeptide is formulated for pharmaceutical administration to a mammal. 
     
     
       17. An antibody, or an antigen binding fragment thereof, which binds specifically to the peptide or polypeptide according to  claim 2 . 
     
     
       18. The antibody or the antigen binding fragment according to  claim 17 , wherein (i) the antibody or antigen binding fragment does not substantially bind to substantially full-length Tau 4R2N trimer polypeptide or (ii) the antibody or antigen binding fragment does not bind to the polypeptide of SEQ ID NO:6. 
     
     
       19. The antibody or the antigen binding fragment according to  claim 17 , wherein the antibody is a monoclonal, polyclonal or monospecific antibody. 
     
     
       20. The antibody or the antigen binding fragment according to  claim 17 , wherein the antibody is humanized or codon-optimized for expression in a mammal. 
     
     
       21. An isolated polynucleotide that encodes the peptide or polypeptide according to  claim 2  or the antibody or antigen binding fragment according to  claim 17 . 
     
     
       22. An expression vector comprising an isolated polynucleotide that encodes the peptide or polypeptide according to  claim 17  or the antibody or antigen binding fragment according to  claim 2 . 
     
     
       23. An isolated host cell transformed with an isolated polynucleotide that encodes the peptide or polypeptide according to  claim 16  or the antibody or antigen binding fragment according to  claim 17 .